DESCRIPTION (Applicant's Description-edited) Breast cancer remains the most common malignant disease of middle-aged women in the United States. Few improvements in the hormonal and cytotoxic therapies of breast cancer have occurred recently. Novel approaches to prevent and treat this disease are required. Recently, studies have shown a dramatic role for all-trans retinoic acid in treatment of several malignant diseases, including acute promyelocytic leukemia. Epidemiologic, in vitro, and animal studies suggest that retinoids and vitamin D3 analogs may inhibit development and/or growth of breast cancer cells. In Specific Aim 1, we will identify and study novel vitamin D3 analogs and retinoids that in vitro can inhibit proliferation and induce differentiation and/or apoptosis of breast cancer cells. We have characterized and have been studying eight breast cancer cell lines in great detail and will use these as our in vitro targets for a large army of novel vitamin D3 and retinoid analogs. Clonogenic analysis will be done as a sensitive assay to determine which analogs can inhibit growth of breast cancer cells. A variety of differentiation assays will be performed to determine how growth is inhibited, whether by cytotoxicity, terminal differentiation, or apoptosis. In addition, the effect of these analogs on invasiveness of breast cancer will be measured in vitro. The retinoids will be characterized as to their affinities for various retinoic acid receptors (RAR alpha, beta, gamma) and the RXR receptors. A third section of our in vitro analysis will endeavor to look at innovative combinations of retinoids and vitamin D analogs. In Specific Aim 2, we will study the abilities of the novel vitamin D analogs and retinoids to inhibit or prevent growth of breast cancer cells in vivo using rodent models. We have been utilizing triple- immunodeficient mice, which allow the easy growth of human breast cancer cell lines. We will test the in vivo efficacy of vitamin D analogs and retinoids that were identified as very active in our in vitro studies. These in vivo studies will allow us to assess toxicity, pharmacology, and antibreast cancer efficacy of these analogs. In addition, we will use a carcinogen-induced breast cancer model in rats to assess the ability of the lead compounds to prevent the development of breast cancer. Specific Aim 3 will take the lead compound(s) identified in our in vitro studies and in vivo animal model studies and test them in patients with breast cancer. The first analog, which we will study in Year Two of this grant, will be 9-cis retinoic acid. Additional lead vitamin D analog(s) will be studied in subsequent years. The in vitro and in vivo studies already accomplished by ourselves and others suggest that the vitamin D analogs and retinoids may offer a novel and creative approach to the management of breast cancer patients. These therapies may be very effective in patients with premalignant breast cancer lesions; those with a high probability of developing additional foci of breast cancer in the contralateral "normal breast"; those who are in remission but have a statistically high probability of relapse. Both vitamin D analogs and retinoids should provide a relatively non- toxic approach to management of these patients. Our project will rapidly and efficiently identify lead compounds, then quickly test them in vivo, initially in animals and subsequently in carefully designed human clinical trials of breast cancer. In addition, our studies should provide insights into the mechanism of action of these unique vitamin D3 analogs and retinoids.